A HUMAN T-CELL RECEPTOR OF THE G-PROTEIN COUPLED RECEPTOR FAMILY

Abstract: This invention relates to nucleotide sequences coding for a human T-cell receptor, to recombinant DNA comprising such nucleotide sequences, vectors comprising such recombinant DNA, and transformed cells and transgenic animals comprising such a nucleotide sequence. This invention further relates to polypeptides which function as a receptor in human T-cells, to antibodies thereto and to ligands which bind to said polypeptide. This invention also relates to an assay for the ability of an agent to act as an agonist or antagonist of said T-cell receptor and to antisense sequences to said nucleotide sequences.

Description: A HUMAN T-CELL RECEPTOR OF THE G-PROTEIN COUPLED RECEPTOR FAMILY , Technical Field This invention relates to a human T-cell receptor. More specifically, this invention relates to nucleotide sequences coding for a human T-cell receptor, to recombinant DNA comprising such nucleotide sequences, vectors comprising such recombinant DNA, and transformed cells and transgenic animals comprising such a nucleotide sequence. This invention further relates to polypeptides which function as a receptor in mammalian cells, to antibodies thereto and to ligands which bind to said polypeptide. Further still, this invention relates to an assay for the ability of an agent to act as an agonist or antagonist of said T-cell receptor. Yet further still, this invention relates to antisense sequences to said nucleotide sequences. Background Art G proteins are well known to those skilled in the art as guanine nucleotide- binding proteins. Hence, G-protein coupled receptors are also well known in the art as receptors which are coupled to guanine nucleotide-binding protein. G proteins transmit signals from the associated receptors which are at the cell-surface to certain effector enzymes within the cell via a GTP binding and hydrolysis cycle. G-protein-coupled receptors typically have seven membrane spanning segments. Chaffin, K. E., et al., EMBO Journal 9:3821-3829 (1990) suggest that a pertussis toxin signalling process involves G ; proteins and regulates thymocyte emigration. Chemofactor response to neutrophil attractant/activation protein-1 (NAP-1 ) by lymphocytes at an efficacy which is low compared to the neutrophil response is reported by Leonard, E. J., et a]., J. Immunol. 144:1323-1330 (1990). The amino acid sequence of NAP-1 has been disclosed by Yoshimura, T., et al., Proc. Natl. Acad. Sci. USA 84:9233-9237 (1987). NAP-1 is also known as interleukin-8 (IL-8). The cloning and sequences of two G-protein coupled somatostatin receptors (SSTR1 and SSTR2) have been disclosed by Yamada, Y., et al., Proc. Natl. Acad. Sci. USA 89:251-255 (1992). The cloning and sequence of the human C5a G-protein coupled receptor have been disclosed by Gerard, N. P., et al., Biochemistry 29:9274- 9281 (1990). The cloning and sequence of a low affinity IL-8 G-protein coupled receptor have been disclosed by Murphy, P. M., et a]., Science 253:1280-1283 (1991) and the cloning and sequence of another IL-8 G-protein coupled receptor have been disclosed by Holmes, W. E., et al., Science 253:1278-1280 (1991). Boulay, F., et a]., Biochemistry 29:11123-11133 (1990) disclose the cloning and sequence of two variants of the human N-formylpeptide chemoattractant G-protein coupled receptor. Polypeptide analogs of an IL-8 receptor-interacting site, antibodies thereto and the use of such antibodies in the immunoassay of IL-8 and to treat inflammation are disclosed in WO9204372, published March 19, 1992. The human complement receptor type 1 gene, the encoded protein and fragments thereof, and uses of such proteins and fragments for treatment of immune disorders, myocardial infarct, damage due to inflammation and, in combination with a thrombolytic, thrombosis are disclosed in WO9105047, published April 18, 1991. The preparation and use of a C5a receptor protein is disclosed in EP377489, published July 11 , 1990. Disclosure of the Invention This invention provides a T-cell receptor and nucleotide sequences encoding the receptor. The nucleotide sequences of this invention consist essentially of the nucleotide sequences shown hereinbelow as SEQUENCE I.D. NO: 1 and SEQUENCE I.D. NO: 5. Notwithstanding the nucleotide sequence presented herein as SEQUENCE I.D. NO: 1 , this invention further provides functional equivalents of that sequence together with functional fragments thereof and functional equivalents of the functional fragments. Included therein is the nucleotide sequence of SEQUENCE I.D. NO: 5, as well as functional equivalents thereof together with functional fragments thereof and functional equivalents of such functional fragments. Preferred are the nucleotide sequences of SEQUENCE I.D. NO: 1 and SEQUENCE I.D. NO: 5. This invention further provides recombinant DNA comprising a nucleotide sequence of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional equivalent of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5. Preferred is such recombinant DNA wherein the nucleotide sequence is functionally linked to a promoter. This invention still further provides vectors comprising recombinant DNA as hereinabove described. Also provided by this invention are transformed or transfected host cells comprising a nucleotide sequence of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional equivalent of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5. The transformed or transfected host cells of this invention are all cells suitable for the replication and/or expression of the nucleotide sequences hereinabove described. By way of example and not of limitation such cells include Escherichia coli, Saccharomvces cerevesiae. Yarrowia lipolvtica and mammalian cells. Yet further still, this invention provides transgenic non-human animals comprising a nucleotide sequence of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional equivalent of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5 or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5. This invention further provides antisense sequences to SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5 as well functional equivalents thereof, functional fragments of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5 and functional equivalents of such functional fragments. The antisense sequences of this invention include all such sequences which are the antisense of the expression sequences of the sequences described above as well as all functional equivalents, functional fragments and functional equivalents of functional fragments of said antisense sequences. The term expression sequence when used in the singular or plural includes, but is not limited to the nucleotide sequences which code for the translational controls and/or the structural sequence. The antisense sequences of this invention are useful to down-regulate the expression of polypeptide SEQUENCE I.D. NO: 2, as well as functional equivalents and fragments thereof as defined below, in cells and, thus, could find usefulness in treating graft rejection and/or inflammatory conditions in an animal, including a human being. Also included in this invention are modified oligonucleotides which bind to said antisense sequences as well as compositions comprising such modified oligonucleotides and antisense sequences. This invention also provides a substantially pure polypeptide of SEQUENCE I.D. NO: 2. Also, provided by this invention are functional equivalents of SEQUENCE I.D. No: 2, functional fragments of SEQUENCE I.D. NO: 2 and functional equivalents of functional fragments of SEQUENCE I.D. NO: 2. The polypeptide of SEQUENCE I.D. NO: 2 is encoded by the nucleotide sequence of SEQUENCE I.D. NO: 5 as well as SEQUENCE I.D. NO: 1 and is a putative human T-cell G-protein coupled receptor . The polypeptides of this invention as hereinabove described are useful for preparing antibodies thereto. Further, the polypeptides are useful in assay procedures such as those described hereinbelow for agonists or antagonists of a T-cell receptor. Still further, the polypeptides of this invention comprising a binding site are useful in the preparation of soluble polypeptides or fragments thereof which inhibit receptor function by competing for binding to the ligand. The polypeptides of this invention are also useful in the isolation of a ligand of the receptor formed by the polypeptide of SEQUENCE I.D. NO: 2 as well as identification of clones expressing a natural ligand thereof from within an expression library. This invention also provides methods to assay an agent for the ability thereof to act as a ligand or an agonist of a T-cell receptor comprising a polypeptide of SEQUENCE I.D. NO: 2, functional equivalents thereof, functional fragments of SEQUENCE I.D. NO: 2 or functional equivalents of functional fragments of SEQUENCE I.D. NO: 2; methods to assay an agent for the ability thereof to act as a selective antagonist of such a T-cell receptor; and methods to assay an agent for the ability thereof to act as an antagonist of such T-cell receptors. The methods are set forth hereinbelow. Also provided by this invention are the antibodies herein described which are useful for identifying cells expressing the receptor formed by the polypeptide of SEQUENCE I.D. NO: 2 and could find usefulness in treating graft rejection and/or inflammatory conditions in an animal, including a human being. Preferably the antibodies are monoclonal antibodies. Still further, this invention provides a ligand to the receptor which ligand is useful in a receptor binding assay to identify a receptor antagonist or agonist. As used throughout this Specification and the appendant claims, "functional fragment" means a fragment of the sequence to which the phrase refers which fragment has a function which is at least part of the overall function of the complete sequence. As used throughout this Specification and the appendant claims, "functional equivalent" when used in the singular or plural means a sequence having the same, or substantially the same function as the sequence to which the phrase refers. As used throughout this Specification and the appendant claims, "recombinant DNA" means DNA which comprises a nucleotide sequence according to this invention regardless of the nature of the DNA. Thus, by way of example and not of limitation, recombinant DNA includes linear DNA fragments as well as chromosomal DNA wherein a nucleotide sequence according to this invention has been inserted as well as derivatives by replication thereof. Description of the Drawing Figure 1 is a map of plasmid pCR11/Da6 wherein pBR322 ori represents the origin of replication from plasmid pBR322, Ampicillin represents the coding sequence for ampicillin resistance, Kanamycin represents the coding sequence for Kanamycin resistance, f 1 ori represents the origin of replication from phage f 1 , Lac Z represents the partial coding regions for β-galactosidase, MCS represents a multiple cloning site, DA6 represents the sequence containing SEQUENCE I.D. NO: 1 as described herein, Hpall represent the restriction site locations for the enzyme Hpall, ATG represents the location of the start codon for the coding sequence of Da6 (i.e., SEQUENCE I.D. NO: 5) and STOP represents the location of the stop codon at the 3' terminus of the coding sequence of Da6 (i.e., SEQUENCE I.D. NO: 5). Detailed Description 1. PREPARATION AND IDENTIFICATION OF CLONE Da6 Employing the guanidium isothiocyanate method described by Chomczynski, P., et a]., Analytical Biochemistry 162: 156-159 (1987), mRNA was isolated from fresh human peripheral blood T cells. According to methods well known to those skilled in the art, and as described by Molecular Cloning. Second Edition, Part 2, Sambrook, J., et a!-. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 8.60-8.63, first strand cDNA was generated from the mRNA using reverse transcriptase. Two primers were commercially prepared by Genosys Corp, Houston, TX. One primer was a degenerate oligonucleotide corresponding to a consensus sequence obtained by comparison of the M2 transmembrane domain of the human interleukin 8 [IL-8, Holmes, W. E., et a}., Science 253:1278-1280 (1991)], F-met-Leu-Phe [FMLP, Boulay, F., et al., Biochemistry 29:11123-11133 (1990)], complement C5a [Gerard, N.P., et a]., Nature 349:614-617 (1991)], neurokinin A [NKA, Gerard, N.P., et aL, The Journal of Biological Chemistry 265:20455-20462 (1990)], and glucocorticoid-induced receptor [MUSGCP, Harrigan, M.T., et a]., Molecular Endocrinology 5:1331-1338 (1991 )]. The M2 degenerate primer was of the sequence ATAAGCTTAANNTNGCNNTNGCNGAC (SEQUENCE I.D. NO: 3). The second primer was a degenerate oligonucleotide corresponding to a consensus sequence obtained by comparison of the M6 transmembrane domain of an IL-8, hFMLP, hC5a, hNKA and MUSGCP receptor. The M6 degenerate primer (reverse complement) was of the sequence TCGAATTCNANNTNNTANGGNNNCCA (SEQUENCE I.D. NO: 4). Using the M2 and M6 degenerate primers of SEQUENCE I.D. NO: 3 and SEQUENCE I.D. NO: 4, the cDNA prepared as described above was amplified using a modified PCR amplification protocol whereby the DNA was amplified 30 cycles with a 30-second denaturization temperature of 94 ° C and a 45-second elongation temperature of 72°C. The annealling temperature was 37°C for the first cycle and the temperature was increased 0.5° C per cycle thereafter. The resulting amplified sequences where cloned into plasmid pBSK (Stratogene, LaJolla, CA) as a restriction fragment (EcoRI/Hindlll). Resulting clones were sequenced using automated fluorescent sequencing on Applied Biosystems 373A Automated DNA sequences (Applied Biosystems Inc. Foster City, CA). A partial Da6 clone was identified by comparison of the translated sequence of the clones to the PIR protein database using BLAST sequence comparison software [National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD; Altschul, S.F., et a]., Journal of Molecular Biology 215:403-410 (1990)]. The partial Da6 clone was then used to isolate the complete Da6 clone by hybridization to a human thymus Λ-gt-11 cDNA library (Clontech, Palo Alto, CA). Initially, about 500,000 plaques, immobilized on nitrocellulose filters, were hybridized to the partial Da6 clone in 5X Denhardt's solution, 5X SSPE, 50% formamide, 100ug/ml ssDNA, 0.1% SDS, and 10% Dextran sulfate at 42°C for 16 hours. The filters then were washed in 0.16X SSC/1.0% SDS at 50° C for 30 minutes. Then, the filters were autoradiographed to X-ray film, with intensifying screens, for 12 hours at -70° C. Resulting positive clones were subjected to two more rounds of plaque screening as described above using the partial Da6 cDNA to purify the full length Da6 clone. A resulting positive clone was amplified and its DNA was recovered as outlined in Molecular Cloning. Second Edition, Part 1 , Sambrook, et al., Op. cjt., pp. 2.69-2.71. Purified DNA was then subjected to PCR (Hoffman LaRoche, Nutley, NJ) using commercially available DNA primers that flank the cloning region of Λgt11 (Clonetech, Palo Alto, CA). The amplified sequences were then cloned into pCRII - TA Cloning System' (Invitrogen, San Diego, CA) using the ligation conditions supplied by the manufacturer. The full length Da6 clone was verified by fluorescent automated sequencing (Applied Biosystems Inc., Foster City, CA). Clone Da6 contains a unique sequence based on searches of both the PIR and Genbank sequence databases (Intelligenetics, Inc., Mountain View, CA). Due to certain homology between Da6 and the platlet activating factor and IL-8 receptors, it was concluded that Da6 encodes a novel putative G-protein coupled receptor. A map of plasmid pCRII/Daδ is presented in Figure 1. Using Da6 as a probe, it was shown by Northern analysis ( Molecular Cloning. Second Edition, Part 1 , Sambrook, J., et a]., Op. cML, pp 7.37-7.52) that the receptor encoded thereby is preferentially expressed in T cells. The nucleotide sequence of the receptor of clone Da6, including its associated 5' and 3' nucleotide sequences, is shown below as SEQUENCE I.D. NO: 1. Clone Da6 which comprises SEQUENCE I.D. NO: 1 in pCRII (Invitrogen, San Diego, CA) has been deposited under the terms of the Budapest Treaty in the American Type Culture Collection, Rockville, MD, a recognized depository affording permanence of the deposits and ready accessibility thereto by the public if a patent is granted on this application. Clone Da6 comprises Escherichia coli transformed with and containing plasmid pCRII/Da6. The Hpall fragment of pCRII/Da6 contains 999bp of Da6 coding sequence (SEQUENCE I.D. NO: 5), 60 bp of Da65' flanking sequence (SEQUENCE I.D. NO: 6), and 554 bp of Da6 3' flanking sequence (SEQUENCE I.D. NO: 7). The deposit is made available during the pendency of this application to one determined by the Commissioner of the United States Patent and Trademark Office to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122, and in accordance with foreign patent laws in countries wherein counterparts of this application, or its progency, are filed. All restrictions on the availability to the public of the microorganism deposited will be irrevocably removed upon granting of the patent. Clone Da6 has been assigned deposit number ATCC 69248. 2. EXPRESSION OF SEQUENCE I.D. NO: 1 IN MAMMALIAN CELLS The full length nucleotide sequence of SEQUENCE I.D. NO: 1 is excised from pCRII/Da6 using restriction enzyme Hpall and is blunt end cloned into the Eco RV restriction sites of vector pcDNA/Neo (Invitrogen, San Diego, CA). For purposes of subsequent antibody recognition , a synthetic flag sequence, CCAGCAGCCATGGACTACAAGGACGACGACGACAAA (SEQUENCE I.D. NO: 8), is inserted at the 5' end of SEQUENCE I.D. NO: 1 in vector pcDNA/Neo by ligation of annealed complementary oligonucleotides containing this sequence in translational frame upstream of the Da6 coding sequence (Harlow, E., et al., Antibodies. 1988, Cold Spring Harbor Laboratory, New York). The vector is then amplified in transformed, competent E. coli cells and purified from those ceils using CsCI gradient purification twice (Molecular Cloning, Second Edition, Part 1 , Sambrook, J., et al., Op. cjt., pp. 1.42-1.43). Appropriate mammalian host cells such as β cells, fibroblasts or COS cells (i.e., cells which do not naturally express a protein of SEQUENCE I.D. NO: 2) are transfected with 10-50 g of the purified vector using calcium chloride, electroporation, liposomes or other means well known to those skilled in art (Molecular Cloning. Second Edition, Part 3, Sambrook, J. et al., Op. cit. , pp. 16.30-16.55). The resulting transfected cells are tested by Northern analysis or reverse transcription polymerase chain reaction (RT-PCR) of isolated mRNA for expression of mRNA corresponding to the flagged polypeptide. Expression of SEQUENCE I.D. NO: 2 from SEQUENCE I.D. NO: 1 is detected using an antibody to the FLAG sequence using FACS, immunoprecipitation, Western blotting or immunohistochemical staining. Such techniques are well known to those skilled in the art and are described in Molecular Cloning. Second Edition, Part 3, Sambrook, J. et al., Op. cjt. pp. 18.19-18.75 and Kunz, D., et a]., J. Biol. Chem. 267: 9101-9106 (1992). Alternatively, an antibody to an extracellular sequence of SEQUENCE I.D. NO: 2 can be used to detect expression according to the techniques described immediately above. 3. PRODUCTION OF ANTIBODIES TO SEQUENCE I.D. NO: 2 Polypeptides corresponding to unique sequences formed within putative extracellular domains of SEQUENCE I.D. NO: 2 [i.e., WFLMYPFRFHDCKQKYDLYI (SEQUENCE I.D. NO: 9) and PLLRTSDDTSGNRTKCFVDLPTRNV (SEQUENCE I.D. NO: 10)] are synthesized and conjugated to an antigen such as key hole limpet hemocyanin (KLH) or bovine serum albumin (BSA). The conjugates are resuspended in an adjuvant and used to immunize and boost animals and sera is drawn after each boost. The sera is tested using an ELISA for the ability to bind the conjugated peptide but not a control peptide. Sera that reacts to the conjugated peptide is then tested for the ability to bind to the surface of SEQUENCE I.D. NO: 2 expressing cells using FACS, ELISA, immunoprecipitation, or Western blotting or immunocytohistochemistry. Alternatively, fusion proteins are prepared using polypeptide sequences from SEQUENCE I.D. NO: 2 and used to immunize animals to produce antisera. To produce such fusion proteins, nucleotide sequences from SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5 can be ligated to lacZ DNA or other DNA and expressed in E. coli. The fusion protein is then purified and used to immunize animals as described above. Alternatively and preferably, monoclonal antibodies to the appropriate extracellular polypeptide sequence are prepared according to standard techniques (Harlow, E., et al, Antibodies, 1988, pp. 55-240, Cold Spring Harbor Laboratory, NY). Polypeptides or fusion proteins containing peptide sequences from SEQUENCE I.D. NO: 2 are used to immunize mice. An appropriate amount of antigen peptides or fusion proteins (5-50 μg) are mixed with complete adjuvant and injected into the intraperitoneal cavity of mice. Alternatively, the antigen may be delivered by subcutaneous injection or by injection directly into lymphoid organs. Subsequent injections are made using incomplete Freund's adjuvant. The sera is tested using an ELISA for the abilty to bind to the appropriate peptide antigen but not a control peptide antigen. Sera that reacts to the appropriate antigen is then tested for the ability to bind to the surface of SEQUENCE I.D. NO: 2 expressing cells using FACS, ELISA, immunoprecipitation or immunocytochemistry. Animals that produce antisera that reacts with the appropriate antigen are used to make hybridomas producing monoclonal antibodies. Animals are boosted with the appropriate antigen 3-5 days prior to fusion. Splenocytes are obtained from the mice and fused to suitable myeloma cells using polyethylene glycol (Harlow, E., et al., Antibodies, Ibid.). The hybridomas are selected for using hypoxanthine, aminoptesin and thymidine Selection (HAT) medium or other suitable selection medium. Hybridoma pools are tested for antibody production using ELISA assay. Positive pools are cloned and individual clones are similarly tested for production of appropriate antibodies. Positive clones are expanded and grown to produce supernatants containing the monoclonal antibody to SEQUENCE I.D. NO: 2. The monoclonal antibodies may be purified on protein A columns (Harlow, E., et al., Antibodies, Op. cjt.) or by other methods known to those skilled in the art of protein purification. 4. DETECTION OF Da6 FUNCTION BY CALCIUM FLUX ASSAY A. Fura-2 assay. SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, transfected cells are incubated with fura-2 acetoxymethyl ester (2.5 ιM) in calcium/magnesium-free PBS for 30 min. at 37°C. Then, the cells are washed, resuspended in calcium/magnesium-free PBS containing 10mM HEPES pH7.4, 0.25% BSA and 10mM glucose. The cells are dispersed into quartz cuvettes and the external calcium concentration is adjusted to 1 mM with CaCI 2 . Receptor ligand is added at an effective concentration and fluorescence is monitored using a fluorescence spectrophotometer at excitation wavelengths of 340 nm and 380 nm and emission wavelength of 510nm. Calcium levels are measured using the ratio of the resulting fluorescence readings. B. Flow cvtometry. According to the method described by Holmes, W. E. et al., Science 253: 1278-1280 (1991), cells transfected with SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, are incubated with indo-1 acetoxymethyl ester (2/ M) in RPMI for 30 min. at 37° C. The entracellular calcium is then measured with a flow cytometer from the ratio of fluorescence at wavelengths of 405 nm and 525 mm. 5. DETECTION OF Da6 FUNCTION BY REPORTER ASSAY Transfection competent cells are co-transfected using electroporation or other transfection protocols (Molecular Cloning, Second Edition, Part 3, Sambrook, J., et al., Op. cit., pp. 16.30-16.55) with a vector capable of expressing SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, in said cells and a vector encoding luciferase under control of a G-protein sensitive promoter (e.g., minimal multimerized CRE, NFkB or minimal multimerized AP-1 promoter driving luciferase as CAT). Minimal multimerized CRE, NFKB or AP-1 promoter luciferase/CAT plasmids are derived by cloning a ligated double strand sequence containing multiple copies of the CRE, NFKB or AP-1 consensus sequence binding sites upstream of a minimal thymidine kinase or IL-2 promoter. The co-transfected cells are stimulated with an effective amount of receptor ligand and functioning of the SEQUENCE I.D. NO: 2 receptor is measured by the amount of luciferase or CAT activity produced by the cell as described in Molecular Cloning, Second Edition, Part 3, Sambrook, J. et a]., Op. cjt., pp. 16.60-16.65, and by DeWet, J.R., et al., Molecular and Cellular Biology 7:725-737 (1987). 6. PROCESS FOR IDENTIFICATION OF NATURAL LIGANDS AND AGONISTS OF SEQUENCE I.D. NO: 2 Employing transfected cells according to the Fura-2, flow cytometry or reporter assay described hereinabove and incubating the cells in the presence of a chemical entity in an appropriate solubilized form, a natural product, a purified factor or a crude cellular extract at varying concentrations and assaying for the effect thereof upon said cells when compared to non-transfected cells enables selection of agonists and natural ligands of SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, as those entities which stimulate calcium flux (Fura-2 or flow cytometry assay) or increase luciferase/CAT activity (reporter assay). 7. IDENTIFICATION OF SELECTIVE SEQUENCE I.D. NO: 2 ANTAGONISTS Cells, stably transfected with and capable of expressing SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, are incubated in the presence of a defined concentration of a chemical entity prior to the addition thereto of a natural ligand or agonist of SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof. In order to assay for selectivity of the antagonist, a known G-protein coupled receptor containing cell line (i.e., an IL-8R expressing control cell line) is also preincubated with the chemical entity prior to addition of a natural ligand (e.g. IL-8) or agonist thereof. In both cases, the effect of the entity on either calcium flux or reporter activity is determined. Those chemical entities which inhibit or block ligand or agonist activation of calcium flux in the stably transfected cells but not in the control cell line are antagonists selective for SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof. Alternatively, a receptor binding assay as described in any one of Kunz, D., et a}., J. Biol. Chem. 267: 9101-9106 (1992), Nourshargh, S., et a]., J. Immunol. 148: 106- 111 (1992), Holmes, W.E., et a]., Science 253: 1278-1280 (1991) or Gerard, N.P., et a]., Biochemistry 29: 9274-9281 (1990) employing a labeled ligand of SEQUENCE I.D. NO:2 can be employed. Transfected cells capable of expressing SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof , a functional fragment thereof or a functional equivalent of a functional fragment thereof, are pre-incubated with varying concentrations of a chemical entity for a pre-determined period of time under appropriate binding conditions followed by the addition thereto of a labeled ligand that binds to SEQUENCE I.D. NO: 2, or a functional equivalent thereof, afunctional fragment thereof or a functional equivalent of a functional fragment thereof. As a control, the binding assay is also performed using the same chemical entity but with a control receptor (e.g. IL-8R) transfected into the same host cell line as was used to obtain the SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, transfected cells and with a labeled ligand of the control receptor. A chemical entity which blocks or inhibits the binding of the labeled ligand to the SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, transfected cells but not control cells is an antagonist selective for SEQUENCE I.D. NO: 2. 8. TRANSGENIC ANIMALS COMPRISING SEQUENCE I.D. NO: 1 OR SEQUENCE I.D. NO: 5 A transgenic vector is constructed comprising SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, together with transcriptional and processing elements to allow expression in the tissue or tissues of choice in the transgenic animal. Expression may be directed to T lymphocytes using vectors described [Cooke, M.P. et a]., Cell 65:281 -291 (1991)]. Once constructed and amplified by methods well known to those skilled in art, approximately 200 copies of linear vector DNA are microinjected into the male pronucleus of the fertilized egg of the animal to be made transgenic according to the methods described by Hanahan, D., Science 246: 1265-1275 (1989) and Brinster, R. L, et a]., The Harvey Lecture Series 80: 1 -38 (1986). The injected eggs are then implanted into the oviduct of pregnant foster mothers and the offspring are tested for transgene integration using dot blot analysis, Southern analysis or PCR analysis as described in Molecular Cloning, Second Edition, Part 2, Sambrook, J., et a]., Op. cjt., pp. 9.4-9.58 and 14.5-14.21. Tissue from animals positive for transgene integration is tested for transgene expression using Northern analysis or PCR analysis for detection of transgene mRNA levels. Transgene positive animals that express the transgene are propagated and the offspring thereof are tested for transgene presence and function to verify establishment of a transgenic animal line. Such transgenic animals are useful for the generation of animal disease models resulting from overexpression of receptors from SEQUENCE I.D. NO: 2. These animals are also useful for testing the effects of antagonists, agonists or natural ligands on receptor function in vivo. 9. ANTISENSE SEQUENCES A synthetic antisense DNA sequence of approximately 15 to 30 base pairs in length and complementary to a portion of an expression sequence of SEQUENCE I.D. NO: 1 , SEQUENCE I.D. NO: 5, a functional equivalent thereof, a functional fragment of SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent of such a functional fragment. The antisense sequence is prepared by standard methods well known to those skilled in the art such as by the use of a DNA synthesizer (Applied Biosystems, Inc., Mountain View, CA). The antisense sequence is then administered to a T-cell or animal at an amount effective to inhibit expression of SEQUENCE I.D. NO: 2, a functional equivalent thereof, a functional fragment of SEQUENCE I.D. NO: 2 or a functional equivalent of such a functional fragment. Further, modified oligonucleotides which bind to the antisense sequences and result in increased cellular uptake, in vivo absorption, bioavailability and/or protection from nucleases can be prepared and employed in compositions with such antisense sequences according to methods described by Cohen, J.S., Pharmac. Ther. 52: 211-225 (1991). SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Hanke, Jeffrey H. Ogborne, Kevin T. Pfizer Inc., (non-U.S.) (ii) TITLE OF INVENTION: HUMAN, RECEPTOR (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Gregg C. Benson, Pfizer Inc (B) STREET: Eastern Point Road (C) CITY: Groton (D) STATE: Connecticut (E) COUNTRY: U.S.A. (F) ZIP: 06340 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Benson, Gregg C. (B) REGISTRATION NUMBER: 30,997 (C) REFERENCE/DOCKET NUMBER: PC8348AGCB (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (203) 441-4901 (B) TELEFAX: (203) 441-5221 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1613 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGGATTGTGA ATTTAGTTTT GAACCATCAT TAGGCAAAGA TAGCTTCTCT AGAGAGAATC 60 ATGCCTGCTA ATTACACGTG TACCAGGCCA GATGGAGACA ATACAGATTT TCGATACTTT 120 ATTTATGCAG TGACATACAC TGTCATTCTT GTGCCAGGTC TCATAGGGAA TATATTAGCC 180 CTGTGGGTAT TCTATGGTTA TATGAAAGAA ACCAAACGAG CTGTGATATT TATGATAAAC 240 TTAGCCATTG CTGACTTACT ACAAGTTCTT TCCTTGCCAC TGAGGATCTT CTACTACTTG 300 AATCATGACT GGCCATTTGG GCCTGGTCTC TGCATGTTCT GTTTCTACCT GAAGTATGTC 360 AACATGTATG CAAGCATCTA CTTCTTGGTC TGCATCAGTG TGCGACGATT TTGGTTTCTC 420 ATGTACCCCT TTCGCTTCCA TGACTGCAAA CAGAAATATG ACCTGTACAT CAGCATTGCT 480 GGCTGGCTGA TCATCTGCCT TGCCTGTGTA CTCTTTCCAC TCCTCAGAAC CAGTGATGAT 540 ACCCCTGGCA ATAGGACCAA ATGCTTTGTG GATCTTCCTA CCAGGAATGT CAACCTGGCC 600 CAGTCCGTTG TTATGATGAC CATTGGCGAG TTGATTGGGT TTGTAACTCC GCTTCTGATT 660 GTCCTATATT GTACCTGGAA GACGGTTTTA TCACTGCAAG ATAAATATCC CATGGCCCAA 720 GATCTTGGAG AGAAACAGAA AGCCTTGAAG ATGATTCTAA CCTGTGCAGG GGTATTCCTA 780 ATTTGCTTTG CACCTTATCA TTTCAGTTTT CCTTTAGATT TCCTGGTGAA GTCCAATGAA 840 ATTAAAAGCT GCCTAGCCAG AAGGGTGATT CTAATATTTC ATTCTGTGGC ATTGTGTCTT 900 GCTAGTCTGA ATTCATGTCT TGACCCAGTC ATATACTACT TTTCCACTAA TGAGTTCCGA 960 AGACGGCTTT CAAGACAAGA TTTGCATGAC AGCATCCAAC TCCATGCAAA ATCCTTTGTG 1020 AGTAACCATA CAGCTTCCAC CATGACACCT GAATTATGCT AAAACAAAAA ACCAAACTGA 1080 ATGTGACCTG AAATGCAAGT ACATCAGAAC ATATCTGCAA TACCCAAGCC ACAGGGAAGA 1140 ACTTGCAAAA CAACACAGCT TTTCAGTTCT GCTCTATCTT ACTGCTATGG GGAATTCACT 1200 TCTTCAAAGC AGGACCTATT TGGAGCATTA CGATCCACGA TTATTGATGT TGACATGTCC 1260 ATGTAGTAAT TTTTCTTCAA GTCTGTAAAT CTTAAAATAT CAAATTTCTG TGACATCCTA 1320 TAAACATATG CACTCAACTG TAGTCAGTAC TTTTACCTGT GAACCTCAGG CACAAAAAGA 1380 TTATTAGCTT GGAGTCACCA TAAACATTTA GTTTTGTTGC AACAGATACT GAGTCTTTAT 1440 GTTCAGAGGA AATGTAAGTG TCTTTTTATA TTAGTAATCA CAGTTTACAT GCCATTTTCA 1500 TATGTTTGGT TATATTTTAG TGGGATAGAT GATATATTAC CCTGTGTAAA GAAAATGTAA 1560 ACATAAGATC ATTTTTATCT CTCAAGTGTG ATTACTCTTC AGAGTTTGAA CCG 1613 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 333 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Pro Ala Asn Tyr Thr Cys Thr Arg Pro Asp Gly Asp Asn Thr Asp 1 5 10 15 Phe Arg Tyr Phe lie Tyr Ala Val Thr Tyr Thr Val lie Leu Val Pro 20 25 30 Gly Leu lie Gly Asn lie Leu Ala Leu Trp Val Phe Tyr Gly Tyr Met 35 40 45 Lys Glu Thr Lys Arg Ala Val lie Phe Met lie Asn Leu Ala lie Ala 50 55 60 Asp Leu Leu Gin Val Leu Ser Leu Pro Leu Arg lie Phe Tyr Tyr Leu 65 70 75 80 Asn His Asp Trp Pro Phe Gly Pro Gly Leu Cys Met Phe Cys Phe Tyr 85 90 95 Leu Lys Tyr Val Asn Met Tyr Ala Ser lie Tyr Phe Leu Val Cys lie 100 105 110 Ser Val Arg Arg Phe Trp Phe Leu Met Tyr Pro Phe Arg Phe His Asp 115 120 125 Cys Lys Gin Lys Tyr Asp Leu Tyr lie Ser lie Ala Gly Trp Leu lie 130 135 140 lie Cys Leu Ala Cys Val Leu Phe Pro Leu Leu Arg Thr Ser Asp Asp 145 150 155 160 Thr Pro Gly Asn Arg Thr Lys Cys Phe Val Asp Leu Pro Thr Arg Asn 165 170 175 Val Asn Leu Ala Gin Ser Val Val Met Met Thr He Gly Glu Leu He 180 185 190 Gly Phe Val Thr Pro Leu Leu He Val Leu Tyr Cys Thr Trp Lys Thr 195 200 205 Val Leu Ser Leu Gin Asp Lys Tyr Pro Met Ala Gin Asp Leu Gly Glu 210 215 220 Lys Gin Lys Ala Leu Lys Met He Leu Thr Cys Ala Gly Val Phe Leu 225 230 235 240 He Cys Phe Ala Pro Tyr His Phe Ser Phe Pro Leu Asp Phe Leu Val 245 250 255 Lys Ser Asn Glu He Lys Ser Cys Leu Ala Arg Arg Val He Leu He 260 265 270 Phe His Ser Val Ala Leu Cys Leu Ala Ser Leu Asn Ser Cys Leu Asp 275 280 285 Pro Val He Tyr Tyr Phe Ser Thr Asn Glu Phe Arg Arg Arg Leu Ser 290 295 300 Arg Gin Asp Leu His Asp Ser He Gin Leu His Ala Lys Ser Phe Val 305 310 315 320 Ser Asn His Thr Ala Ser Thr Met Thr Pro Glu Leu Cys 325 330 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 11 (D) OTHER INFORMATION: /mod_base= OTHER /note= "c or t" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 12 (D) OTHER INFORMATION: /mod_base= OTHER /note= "c or t" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 14 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or a" (ix) FEATURE: (A) NAME/KEY: modifiedjbase (B) LOCATION: 17 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or c" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 18 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g, c or t" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 20 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or a" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 23 (D) OTHER INFORMATION: /mod_base= OTHER /note= "c or t" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATAAGCTTAA NNTNGCNNTN GCNGAC 26 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 9 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or c" (i ) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 11 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or c" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 12 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or c" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 14 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or t" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 15 (D) OTHER INFORMATION: /mod_base= OTHER /note= "a or g" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 18 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or t" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 21 (D) OTHER INFORMATION: /mod_base= OTHER /note= "g or c" (ix) FEATURE: (A) NAME/KEY: modified_base (B) LOCATION: 22 (D) OTHER INFORMATION: /mod_base= OTHER /note= "a or g" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TCGAATTCNA NNTNNTANGG NNCAA 25 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 999 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGCCTGCTA ATTACACGTG TACCAGGCCA GATGGAGACA ATACAGATTT TCGATACTTT 60 ATTTATGCAG TGACATACAC TGTCATTCTT GTGCCAGGTC TCATAGGGAA TATATTAGCC 120 CTGTGGGTAT TCTATGGTTA TATGAAAGAA ACCAAACGAG CTGTGATATT TATGATAAAC 180 TTAGCCATTG CTGACTTACT ACAAGTTCTT TCCTTGCCAC TGAGGATCTT CTACTACTTG 240 AATCATGACT GGCCATTTGG GCCTGGTCTC TGCATGTTCT GTTTCTACCT GAAGTATGTC 300 AACATGTATG CAAGCATCTA CTTCTTGGTC TG.CATCAGTG TGCGACGATT TTGGTTTCTC 360 ATGTACCCCT TTCGCTTCCA TGACTGCAAA CAGAAATATG ACCTGTACAT CAGCATTGCT 420 GGCTGGCTGA TCATCTGCCT TGCCTGTGTA CTCTTTCCAC TCCTCAGAAC CAGTGATGAT 480 ACCCCTGGCA ATAGGACCAA ATGCTTTGTG GATCTTCCTA CCAGGAATGT CAACCTGGCC 540 CAGTCCGTTG TTATGATGAC CATTGGCGAG TTGATTGGGT TTGTAACTCC GCTTCTGATT 600 GTCCTATATT GTACCTGGAA GACGGTTTTA TCACTGCAAG ATAAATATCC CATGGCCCAA 660 GATCTTGGAG AGAAACAGAA AGCCTTGAAG ATGATTCTAA CCTGTGCAGG GGTATTCCTA 720 ATTTGCTTTG CACCTTATCA TTTCAGTTTT CCTTTAGATT TCCTGGTGAA GTCCAATGAA 780 ATTAAAAGCT GCCTAGCCAG AAGGGTGATT CTAATATTTC ATTCTGTGGC ATTGTGTCTT 840 GCTAGTCTGA ATTCATGTCT TGACCCAGTC ATATACTACT TTTCCACTAA TGAGTTCCGA 900 AGACGGCTTT CAAGACAAGA TTTGCATGAC AGCATCCAAC TCCATGCAAA ATCCTTTGTG 960 AGTAACCATA CAGCTTCCAC CATGACACCT GAATTATGC 999 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGGATTGTGA ATTTAGTTTT GAACCATCAT TAGGCAAAGA TAGCTTCTCT AGAGAGAATC 60 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 554 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TAAAACAAAA AACCAAACTG AATGTGACCT GAAATGCAAG TACATCAGAA CATATCTGCA 60 ATACCCAAGC CACAGGGAAG AACTTGCAAA ACAACACAGC TTTTCAGTTC TGCTCTATCT 120 TACTGCTATG GGGAATTCAC TTCTTCAAAG CAGGACCTAT TTGGAGCATT ACGATCCACG 180 ATTATTGATG TTGACATGTC CATGTAGTAA TTTTTCTTCA AGTCTGTAAA TCTTAAAATA 240 TCAAATTTCT GTGACATCCT ATAAACATAT GCACTCAACT GTAGTCAGTA CTTTTACCTG 300 TGAACCTCAG GCACAAAAAG ATTATTAGCT TGGAGTCACC ATAAACATTT AGTTTTGTTG 360 CAACAGATAC TGAGTCTTTA TGTTCAGAGG AAATGTAAGT GTCTTTTTAT ATTAGTAATC 420 ACAGTTTACA TGCCATTTTC ATATGTTTGG TTATATTTTA GTGGGATAGA TGATATATTA 480 CCCTGTGTAA AGAAAATGTA AACATAAGAT CATTTTTATC TCTCAAGTGT GATTACTCTT 540 CAGAGTTTGA ACCG 554 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CCAGCAGCCA TGGACTACAA GGACGACGAC GACAAA 36 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Trp Phe Leu Met Tyr Pro Phe Arg Phe His Asp Cys Lys Gin Lys Tyr 1 5 10 15 Asp Leu Tyr He 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Pro Leu Leu Arg Thr Ser Asp Asp Thr Ser Gly Asn Arg Thr Lys Cys 1 5 10 15 Phe Val Asp Leu Pro Thr Arg Asn Val 20 25

Claims: CLAIMS 1. A nucleotide sequence consisting essentially of SEQUENCE I.D. NO: 1. 2. A functional equivalent of SEQUENCE I.D. NO: 1 , a functional fragment of SEQUENCE I.D. NO: 1 or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 1. 3. Recombinant DNA comprising the nucleotide sequence according to claim 1. 4. Recombinant DNA comprising a nucleotide sequence according to claim 2. 5. Recombinant DNA according to claim 3 wherein the nucleotide sequence is functionally linked to a promoter. 6. Recombinant DNA according to claim 4 wherein the nucleotide sequence is functionally linked to a promoter. 7. A vector comprising recombinant DNA according to claim 3. 8. A vector comprising recombinant DNA according to claim 4. 9. A vector comprising recombinant DNA according to claim 5. 10. A vector comprising recombinant DNA according to claim 6. 11. A transformed or transfected host cell comprising a nucleotide sequence according to claim 1. 12. A transformed or transfected host cell comprising a nucleotide sequence according to claim 2. 13. A nucleotide sequence consisting essentially of SEQUENCE I.D. NO: 5. 14. A functional equivalent of SEQUENCE I.D. NO: 5, a functional fragment of SEQUENCE I.D. NO: 5 or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 5. 15. Recombinant DNA comprising a nucleotide sequence according to claim 13. 16. Recombinant DNA comprising a nucleotide sequence according to claim 14. 17. Recombinant DNA according to claim 15 wherein the nucleotide sequence is functionally linked to a promoter. 18. Recombinant DNA according to claim 16 wherein the nucleotide sequence is functionally linked to a promoter. 19. A vector comprising recombinant DNA according to claim 15. 20. A vector comprising recombinant DNA according to claim 16. 21. A vector comprising recombinant DNA according to claim 17. 22. A vector comprising recombinant DNA according to claim 18. 23. A transformed or transfected host cell comprising a nucleotide sequence according to claim 13. 24. A transformed or transfected host cell comprising a nucleotide sequence according to claim 14 . 25. Escherichia coli ATCC 69248. 26. Plasmid pCRII/Da6. 27. A substantially pure polypeptide of SEQUENCE I.D. NO: 2. 28. A functional equivalent of a polypeptide of SEQUENCE I.D. NO: 2, a functional fragment of SEQUENCE I.D. NO: 2 or a functional equivalent of a functional fragment of SEQUENCE I.D. NO: 2. 29. An antibody to a polypeptide according to claim 27. 30. An antibody to a polypeptide according to claim 28. 31. An antibody according to claim 29 wherein the antibody is a monoclonal antibody. 32. An antibody according to claim 30 wherein the antibody is a monoclonal antibody. 33. A substantially pure ligand which binds to a polypeptide according to claim 27. 34. A substantially pure ligand which binds to a polypeptide according to claim 28. 35. A method to assay an agent for the ability thereof to act as a ligand or an agonist of a cell receptor comprising a polypeptide according to claim 27, a functional equivalent thereof, a functional fragment of a polypeptide according to claim 27, or a functional equivalent of a functional fragment of a polypeptide according to claim 27, which method comprises: (a) incubating cells transfected with and capable of expressing SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, in the presence of a solubilized chemical entity, a natural product, a purified factor or a crude cellular extract; (b) incubating control cells of the same type as in step (a), but which are not so transfected, in the presence of the same solubilized chemical entity, natural product, purified factor or crude cellular extract as employed in step (a); (c) assaying the effect of the solubilized chemical entity, natural product, purified factor or crude cellular extract on the cells of steps (a) and (b); and (d) determining whether the solubilized chemical entity, natural product, purified factor or crude cellular extract increase or stimulate activity of the cell receptor comprising a polypeptide according to claim 27 for the cells of step (a) when compared to the cells of step (b). 36. The method according to claim 35 wherein, in step (c), the effect is assayed by measuring calcium flux. 37. The method according to claim 35 wherein, in step (a), the transfected cells are co-transfected with a vector encoding luciferase under control of a G-protein sensitive promoter; in step (b), the cells are transfected with the vector containing luciferase under control of G-protein sensitive promoter which is used to co-transfect the cells of step (a); and, in step (c), the effect is assayed by measuring luciferase activity. 38. A method to assay an agent for the ability thereof to act as a selective antagonist of a cell receptor comprising a polypeptide according to claim 27, a functional equivalent thereof, a functional fragment of a polypeptide according to claim 27, or a functional equivalent of a functional fragment of a polypeptide according to claim 27, which method comprises: (a) separately incubating cells transfected with and capable of expressing SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, and cells containing a known G-protein coupled receptor in the presence of a chemical entity; (b) adding to the incubations of step (a) a natural ligand or agonist of SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, to the stably transfected cells and a natural ligand or agonist to the known G-protein coupled receptor cells; (c) assaying the effect of the chemical entity on the cells; and (d) determining whether the chemical entity selectively inhibits or blocks the natural ligand or agonist of SEQUENCE I.D. NO: 2, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof. 39. The method according to claim 38 wherein, in step (c), the effect is assayed by measuring calcium flux. 40. The method according to claim 38 wherein, in step (a), the cells are co- transfected and the cells containing a known G-protein coupled receptor are transfected with a vector encoding luciferase under control of a G-protein sensitive promoter; and, in step (c), the effect is assayed by measuring luciferase activity. 41. A method to assay an agent for the ability thereof to act as an antagonist of a cell receptor comprising a polypeptide according to claim 27, a functional equivalent thereof, a functional fragment of a polypeptide according to claim 27, or a functional equivalent of a functional fragment of a polypeptide according to claim 27, which method comprises: (a) separately incubating transfected cells capable of expressing SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, and control cells of the same type but transfected with and capable of expressing a control receptor other than a receptor comprising a polypeptide according to claim 27 with varying concentrations of a chemical entity; (b) adding to the respective cells of step (a) a labeled ligand of the respective receptors thereof; and (c) determining whether the chemical entity blocks or inhibits the binding of the labeled ligand to SEQUENCE I.D. NO: 1 or SEQUENCE I.D. NO: 5, or a functional equivalent thereof, a functional fragment thereof or a functional equivalent of a functional fragment thereof, transfected cells but not to the control cells. 42. A transgenic non-human animal comprising a nucleotide sequence according to claim 1. 43. A transgenic non-human animal comprising a nucleotide sequence according to claim 13. 44. An antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence, or a functional equivalent of a functional fragment of an antisense sequence, wherein the antisense sequence is an antisense sequence to an expression sequence of a nucleotide sequence according to claim 1. 45. An antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence, or a functional equivalent of a functional fragment of an antisense sequence, wherein the antisense sequence is an antisense sequence to an expression sequence of a nucleotide sequence according to claim 2. 46. An antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence, or a functional equivalent of a functional fragment of an antisense sequence, wherein the antisense sequence is an antisense sequence to an expression sequence of a nucleotide sequence according to claim 13. 47. An antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence, or a functional equivalent of a functional fragment of an antisense sequence, wherein the antisense sequence is an antisense sequence to an expression sequence of a nucleotide sequence according to claim 14. 48. A modified oligonucleotide which binds to an antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence or a functional equivalent of a functional fragment of an antisense sequence according to claim 44. 49. A modified oligonucleotide which binds to an antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence or a functional equivalent of a functional fragment of an antisense sequence according to claim 45. 50. A modified oligonucleotide which binds to an antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence or a functional equivalent of a functional fragment of an antisense sequence according to claim 46. 51. A modified oligonucleotide which binds to an antisense sequence, a functional equivalent thereof, a functional fragment of an antisense sequence or a functional equivalent of a functional fragment of an antisense sequence according to claim 47. 52. A composition comprising a modified oligonucleotide according to claim 48 and the antisense sequence, functional equivalent, functional fragment of an antisense sequence or the functional equivalent of a functional fragment of an antisense sequence to which it binds. 53. A composition comprising a modified oligonucleotide according to claim 49 and the antisense sequence, functional equivalent, functional fragment of an antisense sequence or the functional equivalent of a functional fragment of an antisense sequence to which it binds. 54. A composition comprising a modified oligonucleotide according to claim 50 and the antisense sequence, functional equivalent, functional fragment of an antisense sequence or the functional equivalent of a functional fragment of an antisense sequence to which it binds. 55. A composition comprising a modified oligonucleotide according to claim 51 and the antisense sequence, functional equivalent, functional fragment of an antisense sequence or the functional equivalent of a functional fragment of an antisense sequence to which it binds.

Inventors: HANKE JEFFREY HERBERT, OGBORNE KEVIN THOMAS, HANKE, JEFFREY, HERBERT, OGBORNE, KEVIN, THOMAS
Applicants: HANKE JEFFREY HERBERT, OGBORNE KEVIN THOMAS, PFIZER, HANKE, JEFFREY, HERBERT, OGBORNE, KEVIN, THOMAS, PFIZER INC.
Application year: 1994
Main classification: C12N15
IPC codes: A01K67, C07H21, C07K14, C07K16, C12N1, C12N15, C12P21, C12Q1, C12R1, G01N33